In vertebrates, both rod and cone visual pigments consist of an apoprotein, opsin and a chromophore, 11-cis retinal. Upon activation by photons, 11-cis retinal is photo-isomerized to all-trans retinal, which triggers the activation of the phototransduction signaling cascade. Efficient regeneration of 11-cis retinal, through the visual cycle is essential for normal function of photoreceptors. Previous studies suggested that an isomerohydrolase in the RPE is responsible for converting all-trans retinyl ester directly to 11-cis retinol in the visual cycle. Although the isomerohydrolase activity has been known for almost 20 years, the enzyme responsible for this activity has not been identified. RPE65 is a protein predominantly expressed in the RPE and essential for regeneration of 11-cis retinal. A number of mutations in the RPE65 gene are associated with retinal dystrophies. Recent evidence has demonstrated that RPE65 binds to all-trans retinyl ester, the substrate of isomerohydrolase. Our preliminary studies have generated the following new findings about RPE65: 1) Delivery of an adenovirus expressing human RPE65 into the subretinal space of RPE65 knockout (Rpe65 ) mice generated isomerohydrolase activity and partially restored normal retinoid profile in the RPE and retina of Rpe65-/- mice. 2) Two single point mutations, R91W and Y368H from patients with retinal dystrophies abolished the isomerohydrolase activity of RPE65. 3) The isomerohydrolase activity in the RPE is iron-dependent, and RPE65 is an iron-binding protein. 4) Recombinant RPE65, when co-expressed with lecithin:retinol acyl transferase (LRAT) in cultured cell lines, generated robust isomerohydrolase activity in vitro. 5) This isomerohydrolase activity correlated with the expression levels of RPE65 in the cells and is iron-dependent. Based on these findings, we hypothesize that RPE65 is the isomerohydrolase in the visual cycle and is an iron-dependent enzyme. In this project, we will first identify the key amino acid residues in RPE65 for its isomerohydrolase activity. It is our hypothesis that membrane association of RPE65 is essential for its interactions with LRAT and for its isomerohydrolase activity. Therefore, we will determine if the three palmitoylated Cys residues are responsible for membrane anchoring and enzymatic activity of RPE65. We will determine the role of the highly conserved four His and one Glu residues in enzymatic activity of RPE65. Second, we will generate seven point mutations from patients with retinal dystrophies to study their effects on protein stability and enzymatic activity of RPE65 in vitro and in Rpe65-/- mice. Third, we will study the interactions between RPE65 protein and iron and probe the iron binding sites. This project represents a logical extension of our previous studies and will establish RPE65 as the isomerohydrolase and thus, fill the gap in the visual cycle. These studies will contribute to the understanding of the structure and function of this important protein and will also provide new insights into the pathogenesis of retinal dystrophies resulting from RPE65 mutations.